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Method for increasing artemisinin content with FPS (fanesyl pyrophosphate synthase) and DBR2 (artemisinic aldehyde Delta11(13) reductase) genes for co-transformation and prepared artemisinin

A transgenic and artemisinin technology, applied in the field of genetic engineering, can solve problems such as difficult maintenance, high equipment cost, and limited output, and achieve the effect of enhancing metabolic pathways

Active Publication Date: 2017-05-31
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although a major breakthrough has been made through the chemical semi-synthesis of artemisinin through synthetic biology, the multi-step key enzyme genes in the artemisinin biosynthetic pathway have been introduced into the yeast genome, and high-yield artemisinic acid and artemisinic acid have been successfully obtained in yeast. Artemisinin is then synthesized in vitro by chemical methods. This method is successful in the laboratory stage, but the semi-synthetic equipment through yeast fermentation is costly in the early stage, difficult to maintain, and the output is limited, which cannot fully meet the market demand.

Method used

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  • Method for increasing artemisinin content with FPS (fanesyl pyrophosphate synthase) and DBR2 (artemisinic aldehyde Delta11(13) reductase) genes for co-transformation and prepared artemisinin
  • Method for increasing artemisinin content with FPS (fanesyl pyrophosphate synthase) and DBR2 (artemisinic aldehyde Delta11(13) reductase) genes for co-transformation and prepared artemisinin
  • Method for increasing artemisinin content with FPS (fanesyl pyrophosphate synthase) and DBR2 (artemisinic aldehyde Delta11(13) reductase) genes for co-transformation and prepared artemisinin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, cloning of Artemisia annua FPS and DBR2 gene

[0028] Farnesyl pyrophosphate synthase (FPS) is the key enzyme that catalyzes the condensation of IPP and DMAPP to generate FPP. FPP is the common precursor of various terpenoids in plants and the precursor of artemisinin. DBR2 enzyme catalyzes artemisinic aldehyde to dihydroartemisinic aldehyde.

[0029] 1. Extraction of Total RNA from Artemisia annua Genome

[0030] Take the leaf tissue of Artemisia annua, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf (EP) centrifuge tube filled with lysate, shake it fully, and extract total RNA according to the instructions of the TIANGEN kit. The quality of total RNA was identified by agarose gel electrophoresis, and then the RNA concentration and purity were determined on a spectrophotometer.

[0031] 2. Cloning of Artemisia annua FPS and DBR2 genes

[0032] With the extracted total RNA as a template, cDNA is synthesized under the effect of PowerScript rev...

Embodiment 2

[0034] Embodiment 2, the construction of the plant expression vector containing FPS and DBR2 gene

[0035] 1. Construction of intermediate vectors pMD18T-FPS and pMD18T-DBR2

[0036] The pMD18T vector (Takara, Dalian) was selected as the cloning vector to construct intermediate vectors pMD18T-FPS and pMD18T-DBR2. Specifically, the full-length genes of FPS and DBR2 were respectively amplified by using high-fidelity enzymes, and the primer sequences are shown in Table 1. At the same time, SpeI and BstEII restriction sites were introduced on both sides of FPS and DBR2 genes, respectively. The full-length gene with restriction sites was ligated to the pMD18T vector by T4 ligase, and the sequencing was completed by Shanghai Meiji Biomedical Technology Co., Ltd.

[0037] 2. Construction of plant gene expression cassettes CaMV 35S-FPS-nos and CaMV 35S-DBR2-nos

[0038]Using pCAMBIA1304 as the expression vector, connect FPS and DBR2 into their corresponding restriction sites. Sp...

Embodiment 3

[0046] Example 3, Agrobacterium tumefaciens mediated genetic transformation of FPS and DBR2 genes to obtain transgenic plants of Artemisia annua

[0047] 1. Acquisition of plant expression vector Agrobacterium tumefaciens containing FPS and DBR2 genes

[0048] The plant expression vector containing FPS and DBR2 in Example 2 was transformed into Agrobacterium tumefaciens by freeze-thaw method (such as EHA105, which is a publicly available biological material in the market, which can be purchased from CAMBIA Company in Australia, and the strain number is Gambar 1) , and carry out PCR verification, the verification result is as follows figure 2 shown. The results showed that the plant expression vector containing FPS and DBR2 had been successfully transformed into the Agrobacterium tumefaciens strain.

[0049] When using the freeze-thaw method to transfer the plant expression vector into Agrobacterium tumefaciens, add the plant expression vector to the competent cells of Agr...

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Abstract

The invention discloses a method for increasing the artemisinin content in southernwood, which co-transforms southernwood with FPS (fanesyl pyrophosphate synthase) gene and DBR2 (artemisinic aldehyde Delta11(13) reductase) gene. The artemisinin content in southernwood is determined by a high-performance liquid chromatography-evaporative light scattering detector, and a genetically modified southernwood plant with increased artemisinin content is obtained by screening. The content of artemisinin in the genetically modified southernwood obtained by the invention is remarkably increased, reaching 3.36 times as high as the content of artemisinin in a non-transformed control plant at most.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an Artemisia annua co-transformed with two key enzyme genes FPS and DBR2 and a preparation method thereof. The Artemisia annua has increased artemisinin content. Background technique [0002] Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin, a sesquiterpene lactone compound containing a peroxy bridge extracted from its aerial part, is currently the most widely used and effective antimalarial drug, especially for cerebral malaria and anti-chloroquine malaria. Currently, artemisinin combination therapies (ACTs) are the most effective treatment for malaria recommended by the World Health Organization. However, the content of artemisinin in the plant Artemisia annua is very low, which cannot fully meet the global market demand. Artemisia annua has secretory glandular trichomes and non-glandular trichomes. There are a large number of ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66A01H5/00
CPCC12N9/1085C12N15/66C12N15/8205C12N15/8243C12Y205/01001C12Y205/0101
Inventor 唐克轩石璞付雪晴沈乾江伟民马亚男郝小龙孙小芬
Owner SHANGHAI JIAO TONG UNIV
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